Methods and compositions for treatment of myotonia

The present application is a U.S. national stage application based on PCT/US05/007631 entitled Methods and Compositions for Treatment of Diseases Associated with Aberrant Microsatellite Expansion, filed Mar. 10, 2005, which claims priority to U.S. Provisional Application Ser. No. 60/551,748, filed on Mar. 10, 2004,the entire contents of each of which is incorporated herein by reference.

Each of the applications and patents cited in this text, as well as each document or reference cited in each of the applications and patents (including during the prosecution of each issued patent; “application cited documents”), and each of the PCT and foreign applications or patents corresponding to and/or claiming priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference, and may be employed in the practice of the invention. More generally, documents or references are cited in this text, either in a Reference List before the claims, or in the text itself; and, each of these documents or references (“herein cited references”), as well as each document or reference cited in each of the herein cited references (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference.

This work was supported in part by a grant from the National Institutes of Health (U54-NS48843). The United States Government has certain rights to the invention

Microsatellite Expansion Diseases

Aberrant expansion of microsatellites in DNA is associated with a number of neurological and neuromuscular diseases (O'Donnell, W T, Warren, S T (2002), Annu. Rev. Neurosci. 25: 315). These diseases are caused by microsatellite repeat expansions in coding and non-coding regions. The characterized coding region expansion diseases include Dentatorubral pallidoluysian atrophy (DRPLA), Huntington chorea (HD), Oculopharyngeal muscular dystrophy (OPMD), Spinobulbar muscular atrophy (SBMA), and Spinocerebellar ataxia types 1, 2, 3, 6, 7, and 17 (SCA1, SCA2, SCA3, SCA6, SCA7, SCA17). The characterized non-coding region expansion diseases include Fragile XA, Fragile XE, Friedrich's ataxia, Myotonic Dystrophy type 1 (DM1), Myotonic Dystrophy type 2 (DM2), and Spinocerebellar ataxia types 8, 10, and 12 (SCA8, SCA10, SCA12). Huntington's disease-like type 2 (HDL2) is likewise caused by a microsatellite expansion.

Microsatellite expansion diseases have been most commonly associated with trinucleotide expansion mutations. In fact, at least 16 of the microsatellite expansion diseases reported to date have been characterized as trinucleotide expansion diseases. More recently, however, microsatellite expansion diseases have also been associated with tetranucleotide and even pentanucleotide expansion mutations. Disease severity and age of onset have both been related to the size of the expansion mutation, eventually leading to muscle weakness and premature cataract formation, and, in severe cases, to hypotonia, muscle heart block, and nervous system dysfunction (Korade-Mirnics, Z, Babitzke, P, Hoffman, E (1998) Nuc. Acids Res. 26(6): 1363-1368).

Myotonic dystrophy (dystrophia myotonica, DM) is a multisystemic, dominantly inherited disorder often characterized by myotonia, or, delayed muscle relaxation due to repetitive action potentials in myofibers, and muscle degeneration. Manifestations of DM may also include heart block, ocular cataracts, hypogonadism, and nervous system dysfunction.

Myotonic dystrophy type 1 (DM1) is caused by a trinucleotide (CTG) n expansion (n=50 to >3000) in the 3′-untranslated region (3′UTR) of the Dystrophia myotonica-protein kinase (DMPK) gene. Myotonic dystrophy type 2 (DM2) is caused by a tetranucleotide (CCTG) n expansion (n=75 to ˜11,000) in the first intron of zinc finger protein 9 (ZNF9) gene (Ranum, L P W, Day, J W (2002) Curr. Opin. in Genet. and Dev. 12:266-271).

Although the expansions are located on different chromosomes, there appears to be a common pathogenic mechanism involving the accumulation of transcripts into discrete nuclear RNA foci containing long tracts of CUG or CCUG repeats expressed from the expanded allele (Liquori C L, Ricker K, Moseley M L, Jacobsen J F, Kress W, Naylor S L, Day J W, Ranum L P (2001), Science 293: 864-867).

In effect, both DM1 and DM2 mutant transcripts accumulate as foci within muscle nuclei (Liquori, et al., 2001). An indication that these transcripts are pathogenic comes from studies on HSA LR mice, which express a large CTG repeat in the 3′-UTR of a human skeletal actin transgene (Mankodi, A, Logigian, E, Callahan, L, McClain, C, White, R, Henderson, D, Krym, M, Thornton, C A (2000) Science 289: 1769-1773). These transgenic mice develop myonuclear RNA foci, myotonia, and degenerative muscle changes similar to those seen in human DM. The myotonia in HSA LR mice is caused by loss of skeletal muscle chloride (ClC-1) channels due to aberrant pre-mRNA splicing (Mankodi, A, Takahashi, M P, Jiang, H, Beck, C L, Bowers, W J, Moxley, R T, Cannon, S C, Thornton, C A (2002) Mol. Cell 10: 35-44). Similar ClC-1 splicing defects exist in DM1 and DM2. However, the connection between accumulation of mutant DM transcripts in the nucleus and altered splice site selection has not been established (Faustino, N A, Cooper, T A (2003) Genes Dev. 17: 419-437).

The RNA gain-of-function hypothesis proposes that mutant DM transcripts alter the function and localization of alternative splicing regulators, which are critical for normal RNA processing. Consistent with this proposal, misregulated alternative splicing in DM1 has been demonstrated for six pre-mRNAs: cardiac troponin T (cTNT), insulin receptor (IR), muscle-specific chloride channel (ClC-1), tau, myotubularin-related protein 1 (MTMR1) and fast skeletal troponin T (TNNT3) (Kanadia R N, Johnstone K A, Mankodi A, Lungu C, Thornton C A, Esson D, Timmers A M, Hauswirth W W, Swanson M S (2003), Science 302: 1978-1980).

In all cases, normal mRNA splice variants are produced, but the normal developmental splicing pattern is disrupted, resulting in expression of fetal protein isoforms that are inappropriate for adult tissues. The insulin resistance and myotonia observed in DM1 correlate with the disruption of splicing of two pre-mRNA targets, IR and ClC-1, respectively (Savkur R S, Philips A V, Cooper T A, Dalton J C, Moseley M L, Ranum L P, Day J W (2004), Am J Hum Genet 74:1309-1313).

The mechanism by which expanded repeats alter the regulation of pre-mRNA alternative splicing is unclear. Two families of RNA-binding proteins have been implicated in DM1 pathogenesis: CUG-BP1 and ETR-3-like factors (CELF) and muscleblind-like (MBNL) proteins (Ladd A N, Charlet-B N, Cooper T A (2001), Mol Cell Biol 21: 1285-1296). Six CELF (also called BRUNOL) genes have been identified in humans (Ladd A N, Nguyen N H, Malhotra K, Cooper T A (2004), J Biol Chem 279: 17756-17764). All six CELF proteins have been shown to regulate pre-mRNA alternative splicing and two (CUG-BP1 and ETR-3/CUG-BP2) have been shown to have cytoplasmic RNA-associated functions (Mukhopadhyay D, Houchen C W, Kennedy S, Dieckgraefe B K, Anant S (2003), Mol Cell 11: 113-126).

A functional link has been established between splicing regulation by CELF proteins and DM1 pathogenesis. CUG-BP1 regulates alternative splicing of at least three of the pre-mRNAs (cTNT, IR and ClC-1) that are misregulated in DM striated muscle (Charlet-B N, Savkur R S, Singh G, Philips A V, Grice E A, Cooper T A (2002b), Mol Cell 10: 45-53). The splicing patterns observed for all three pre-mRNAs are consistent with increased CUG-BP1 activity and an increase in CUG-BP1 steady-state levels in DM1 striated muscle (Charlet-B N, Savkur R S, Singh G, Philips A V, Grice E A, Cooper T A (2002b), Mol Cell 10: 45-53).

Furthermore, cTNT minigenes expressed in DM1 muscle cultures or cTNT and IR pre-mRNAs co-expressed with CUG repeat RNA in normal cells reproduce the aberrant splicing patterns observed for endogenous genes in DM cells (Philips A V, Timchenko L T, Cooper T A (1998), Science 280: 737-741; Savkur R S, Philips A V, Cooper T A (2001), Nat Genet 29: 40-47). The trans-dominant effects of endogenous or co-expressed CUG repeat RNA on cTNT and IR splicing regulation require the intronic CUG-BP1-binding sites, indicating that binding by CUG-BP1 and/or other CELF family members to their cognate intronic regulatory elements is required for induction of aberrant splicing regulation by CUG repeat RNA (Philips A V, Timchenko L T, Cooper T A (1998), Science 280: 737-741; Savkur R S, Philips A V, Cooper T A (2001), Nat Genet 29: 40-47).

The CNS symptoms of DM1 may include cognitive impairment, hypersomnolence, heightened sensitivity to anesthetic agents, central hypoventilation, neuroendocrine dysfunction, and effects on personality and behavior [reviewed by Harper (Harper, P. S. (2001), Myotonic dystrophy . Saunders London) and Ashizawa (Ashizawa, T. (1998), Arch. Neurol., 55, 291-293)]. Some of these effects, such as, mental retardation in individuals with congenital DM1, occur during development (Dyken, P. R., Harper, P. S. (1973), Neurology, 23, 465-473). Other symptoms, such as, hypersomnolence, appear during adult life. The mechanism and neuropathologic correlates for CNS involvement in DM1 are unknown.

It is presently unclear whether any steps in the pathogenic sequence of poly(CUG) expression, formation of RNA inclusions, sequestration of RNA binding proteins, and disruption of alternative splicing can take place in the CNS. There is controversy about which cells in the mature brain, if any, express DMPK (Lam, L. T., Pham, Y. C., Nguyen, T. M., and Morris, G. E. (2000), Hum. Mol. Genet., 9, 2167-2173).

Microtubule-associated protein tau (MAPT) pre-mRNA is alternatively spliced at exons 2, 3, and 10 (Goedert, M., Spillantini, M. G., Jakes, R., Rutherford, D., and Crowther, R. A. (1989), Neuron, 3, 519-526). Tau transcripts in fetal brain do not include exon 10, whereas ˜50% of transcripts in adult brain include this exon which encodes an additional microtubule binding domain (Hong, M., Zhukareva, V., Vogelsberg-Ragaglia, V., Wszokek, Z., Reed, L., Miller, B. I., Geschwind, D. H., Bird, T. D., McKeel, D., Goate, A. et al. (1998), Science, 282, 1914-1917). Alternative splicing of exons 2 and 3 also is developmentally regulated (neither exon is included in the fetus, adults mainly include exon 2).

The relative proportion of tau splice products is tightly regulated, as shown by kindreds with frontotemporal dementia and parkinsonism (FTDP-17) due to mutations in MAPT. Silent mutations in MAPT exon 10, or, in the flanking intron, lead to FTDP-17 by disrupting cis elements that regulate splicing of tau pre-mRNA (D'Souza, I., Poorkaj, P., Hong, M., Nochlin, D., Lee, V. M., Bird, T. D., and Schellenberg, G. D. (1999), Proc. Natl. Acad. Sci. U.S.A, 96, 5598-5603). Usually these mutations lead to increased inclusion of exon 10 (Lee, V. M., Goedert, M., and Trojanowski, J. Q. (2001), Annu. Rev. Neurosci., 24, 1121-1159). However, some MAPT mutations that segregate with FTDP-17 have the opposite effect of reducing exon 10 inclusion (Stanford, P. M., Shepherd, C. E., Halliday, G. M., Brooks, W. S., Schofield, P. W., Brodaty, H., Martins, R. N., Kwok, J. B., and Schofield, P. R. (2003), Brain, 126, 814-826).

RNA-binding proteins that regulate alternative splicing bind to sequence-specific elements in the pre-mRNA to enhance or repress inclusion of alternative exons. Aberrant regulation of alternative splicing can cause the expression of inappropriate splicing patterns leading to human disease (Faustino and Cooper, 2003). Myotonic dystrophy constitutes an example of a disease that alters the function of RNA-binding proteins to cause misregulated alternative splicing.

The present disclosure provides methods and compositions for treating diseases associated with aberrant microsatellite expansion employing recombinant adeno-associated virus (rAAV) expressing human muscleblind (MBNL) proteins.

One embodiment of the invention is directed to a method of treating a disease associated with aberrant microsatellite expansion, comprising administering to a mammal in need thereof, a therapeutically effective amount of recombinant adeno-associated virus (rAAV) containing a transgene that encodes a protein selected from the group consisting of MBNL1, MBNL2, MBNL3, and combinations thereof. In one embodiment of the invention, treating comprises ameliorating or eliminating the symptoms of a neuromuscular or neurological condition caused by the aberrant microsatellite expansion. In an additional embodiment of the invention, the neuromuscular condition is myotonic dystrophy.

In other embodiments of the invention, treating comprises reversing the mis-splicing of the Clcn1 skeletal muscle chloride channel, reversing the mis-splicing of the Amyloid beta (A4) precursor protein (APP), reversing the mis-splicing of the NMDA receptor NR1 (GRIN1), reversing the mis-splicing of the Microtubule-associated protein tau (MAPT), or reversing the mis-splicing of TNNT2 (cTNT), respectively.

One embodiment of the invention is directed to a method of treating a disease associated with aberrant microsatellite expansion, comprising administering to a mammal in need thereof, a therapeutically effective amount of recombinant adeno-associated virus (rAAV) containing a transgene that encodes MBNL1.

One embodiment of the invention is directed to a method of treating a disease associated with aberrant microsatellite expansion, comprising administering to a mammal in need thereof, a therapeutically effective amount of recombinant adeno-associated virus (rAAV) containing a transgene that encodes a protein selected from the group consisting of MBNL1, MBNL2, MBNL3, and combinations thereof, wherein the mammal is human. In another embodiment of the invention, the mammal in need of treatment has RNA inclusions in neuronal cells.

One embodiment of the invention is directed to pharmaceutical compositions comprising a recombinant adeno-associated virus (rAAV) containing a transgene that encodes at least one protein selected from the group consisting of MBNL1, MBNL2, MBNL3, and combinations thereof. In another embodiment of the invention, the protein is MBNL1.

The present disclosure also provides a mouse model for myotonic dystrophy, wherein the mouse has a substantial deletion of a muscleblind exon in its genome. Such an animal model for human disease allows the identification and testing of potential therapeutic and preventive agents.

Accordingly, one embodiment of the invention is directed to a mouse model for disease associated with aberrant microsatellite expansion, comprising a mouse having a substantial deletion of Mbnl1 exon 3 (E3) in the mouse genome, wherein said mouse exhibits symptoms typical of a disease associated with aberrant microsatellite expansion in humans. In another embodiment, the invention is directed to a cell isolated from said mouse. In one embodiment of the invention, the mouse exhibits symptoms such as muscle weakness and ocular cataracts.

In one embodiment, the invention is directed to a mouse model for disease associated with aberrant microsatellite expansion, comprising a mouse having a substantial deletion of Mbnl1 exon 3 (E3) in the mouse genome, wherein said mouse exhibits symptoms typical of a disease associated with aberrant microsatellite expansion in humans, wherein the microsatellite repeat expansion disease is caused by a microsatellite expansion in a coding region of DNA. In another embodiment of the invention, the microsatellite repeat expansion disease is caused by a microsatellite expansion in a non-coding region of DNA.

In one embodiment, the invention is directed to a mouse model for disease associated with aberrant microsatellite expansion, comprising a mouse having a substantial deletion of Mbnl1 exon 3 (E3) in the mouse genome, wherein said mouse exhibits symptoms typical of a disease associated with aberrant microsatellite expansion in humans, wherein the mouse exhibits abnormal muscleblind proteins. In other embodiments of the invention, the mouse may have a loss of functional ClC-1 protein, a loss of functional Amyloid beta (A4) precursor protein, a loss of functional NMDA receptor NR1, a loss of functional Microtubule-associated protein tau, a loss of functional TNNT2 protein, or a loss of functional TNNT3 protein, respectively.

One embodiment of the invention is directed to a method of identifying a compound useful in the treatment of disease associated with aberrant microsatellite expansion, comprising administering a test compound to a mouse having a substantial deletion of Mbnl1 exon 3 (E3) in the mouse genome, wherein said mouse exhibits symptoms typical of a disease associated with aberrant microsatellite expansion in humans, wherein the mouse exhibits abnormal muscleblind proteins, and monitoring said mouse for reduction or inhibition of the symptoms associated with said disease. In an additional embodiment, the mouse may be monitored for effects other than those associated with the disease. In one embodiment of the invention, the disease is myotonic dystrophy.

FIG. 1A shows targeted disruption of Mbnl1. The illustration includes C57BL/6J Mbnl1 exon organization (open boxes, UTRs black boxes, open reading frame) together with the 129S1/Svlm] insert (black rectangle), the 129 genomic region with EcoRV (E) (E site in C57BL/6] shown by black box with white E), Xba1 (X), and Bam HI (B) sites, the targeting construct with a thymidine kinase marker (TK), floxed (black triangles, loxP sites), neomycin cassette (stippled box with white N), the 129 region (thick black line) and locations of hybridization probes I and II.

FIG. 1B is a genomic analysis of Mbnl1 mice with the use of probe 1. The 11-kb EcoRV fragment is derived from C57BL/6; the mutant is 6.5 kb.

FIG. 1C shows loss of Mbnl1 E3 expression in Mbnl1 ΔE3/ΔE3

FIG. 1D is an immunoblot analysis (total spleen protein) showing absence of Mbnl1 41-42 kD proteins in Mbnl1 ΔE3/ΔE3 .

FIG. 2A shows an electromyograph (EMG) of Mbnl1 wild-type and mutant knockout vastus muscle. The arrow (top panel) indicates normal EMG electrode insertional activity in wild-type muscle, whereas insertion triggers myotonic discharges in Mbnl1 ΔE3/ΔE3 muscle (bottom panel).

FIG. 2B shows ClC-1 splicing in DM mouse models. Functional chloride channels are produced when Clcn1 exons 6, 7 and 8 are spliced directly together, whereas isoforms that include cryptic exons 7a or 8a encode truncated non-functional proteins. Clcn1 exons 7 to 8 are illustrated (open boxes) with the primer positions indicated via horizontal arrows. Inclusion of exons 7a and 8a occurs at low levels in wild-type (FVB wt, Mbnl1 +/+ ) and Mbnl1 +/ΔE3 muscle but at increased levels in Mbnl1 ΔE3/ΔE3 and HSA LR muscle.

FIG. 2C and FIG. 2D depict the loss of ClC-1 protein observed in Mbnl1 ΔE3/ΔE3 vastus muscle. Representative images of sections from 11-week-old mice show reduced ClC-1 immunostaining in Mbnl1 ΔE3/ΔE3 mice (D) relative to wild-type mice (C). Scale bar, 20 μm.

FIG. 2E and FIG. 2F constitute representative images of sections from 11-week-old mice showing equivalent dystrophin (Dys) levels in Mbnl1 +/+ (E) and Mbnl1 ΔE3/ΔE3 (F) muscle.

FIG. 2G and FIG. 2H depict abnormal muscle histology. Hematoxylin and eosin (H&E)-stained vastus from wild-type (G) and Mbnl1 ΔE3/ΔE3 (H) mice, showing split myofibers (black arrowhead) and centralized myonuclei (white arrowhead). Scale bar, 30 μm.

FIG. 2I to FIG. 2L show cataract development. Dilated eyes of 18-week old mice showing a clear wild-type lens (I) but dust-like opacities (white arrowhead) in Mbnl1 ΔE3/ΔE3 mice (K). Center bright spot is the lamp reflection. H&E-stained anterior section (J, L) highlight increased fragmentation (black arrowhead) and opacities (white arrowhead) in Mbnl1 ΔE3/ΔE3 lens (L) compared to wild-type lens (J).

FIGS. 3A and 3B constitute representative images of sections from 11-week-old mice showing similar levels of α-sarcoglycan in (A) wild-type (Mbnl1 +/+ ) vastus muscle and (B) muscleblind E3 knockout (Mbnl1 ΔE3/ΔE3 ) vastus muscle.

FIG. 4A shows adult retention of Tnnt2 exon 5 Mbnl1 ΔE3/ΔE3 heart. RT-PCR products with (+) and without (−) exon 5 (black box) are indicated (brackets). Size markers are pBR322 Msp I fragments.

FIG. 4B shows Tnnt3 fetal (F) exon inclusion in adult Mbnl1 ΔE3/ΔE3 . The Tnnt3 protein contains variable N-terminal (alternative splicing of exons 4 to 8 and F) and C-terminal regions (exons 16 and 17) (23). RT-PCR (11-week-old mice) of Tnnt3 exons 2 to 11 (left panel) is shown with alternatively spliced exons 4 to 8 and the fetal (F) exon (black boxes). The F exon contains a BsrBI site (arrowhead) resulting in co-migrating smaller fragments in Mbnl1 ΔE3/ΔE3 (right panel).

FIG. 4C depicts RT-PCR of Tnnt3 exons 15 to 18 after MscI digestion.

FIG. 4D shows retention of Tnnt3 fetal (F) exon in adult DM1 skeletal muscle (left panel). The right panel shows cDNAs containing the F exon (bracket) cleaved with BbsI (arrowhead).

FIG. 5A shows reversal of the skeletal muscle major chloride channel (Clcn1) splicing defect following AAV-MBNL1 injection. +lanes represent AAV-mycMBNL1 injection into the Tibialis anterior (TA) muscles of HSA LR mice, while −lanes represent injection of PBS into the other leg. Boxes indicate Clcn1 exons. Shown are the normal (bottom, exons 6, 7, 8 spliced directly together) and aberrant (7a, 8a and intron 6 inclusion) splicing patterns. Mice 190 and 191 are uninjected controls.

FIG. 5B shows an electromyogram depicting the results of the myotonia analysis performed. The scale (Y-axis) runs from 0 to 3, with 3 corresponding to severe myotonia. Zero equals no observed myotonic discharges, 1 equals occasional myotonic discharge, 2 equals abundant myotonic discharges and 3 equals myotonic discharge in nearly every insertion. The X-axis shows the mouse number and whether the TA was injected or uninjected with rAAV1/Myc-hMBNL1.

FIG. 6 shows the results of RT-PCR analysis of exon inclusion. Percent exon inclusion is calculated as ((mRNA+exon)/(mRNA−exon+mRNA+exon))×100. Results are derived from at least three independent experiments. Expression of GFP-MBNL1 (˜72 kDa), GFP-MBNL2 (˜58 kDa), GFP-MBNL3 (˜70 kDa) and EGFP (˜27 kDa) was detected by Western blot analysis using an anti-GFP monoclonal antibody. All three MBNL proteins promote exon 5 skipping of (A) chicken and (B) human cTNT exon 5 in primary skeletal muscle cultures. (C) All three MBNL proteins promote exon 11 inclusion in a human IR minigene in HEK293 cells. (D) MBNL proteins have minimal effects on splicing of exon EN in a clathrin light-chain B minigene in primary skeletal muscle cultures.

FIG. 7A shows a Western blot confirming depletion of endogenous MBNL1 by independent transfection of two different siRNA constructs using the MBNL1 monoclonal (mAb) 3A4, which recognizes two MBNL1 isoforms generated by alternative splicing (˜41 and 42 kDa). GAPDH (˜36 kDa) was used as a loading control.

FIG. 7B shows the results of immunofluorescence analysis using mAb 3A4 to confirm depletion of endogenous protein after independent transfection of each MBNL1 siRNA construct. Scale bar, 10 μm.

FIG. 7C shows, in bar graph form, the RT-PCR results from at least three transfections.

FIG. 8 shows MBNL1 binds upstream of exon 5 in human cTNT at a site distinct from the CUG-BP1-binding site. (A) Binding of recombinant GST-MBNL1 to uniformly 32 P-labeled RNA was assayed by UV cross-linking. Scanning mutagenesis was performed by replacing 6 nt blocks with AUAAUA and identified two binding sites 18 and 36 nt upstream of the alternative exon. The MBNL1-binding sites (M) and the CUG-BP1-binding site (C) are located on opposite sides of exon 5. (+) and (−) indicate binding; (·)indicates a putative branch point adenosine. (B) Four nucleotide substitutions significantly reduce binding of recombinant MBNL1 detected by UV cross-linking. Competition of GST-MBNL1 binding to 32 P-labeled RNA G by the indicated picomoles of non-labeled RNAs G or M shown in A). (C, D) MBNL1-binding site mutations reduce responsiveness to MBNL1, MBNL2 and MBNL3 co-expression but not CUG-BP1 in COSM6 cells. Human cTNT minigenes containing the natural sequence (C) or the four nucleotide substitutions (mutation M in A) in the MBNL1-binding site (D) were co-expressed with GFP or the indicated GFP fusion proteins. Exon inclusion was assayed by RT-PCR.

FIG. 9A schematically depicts how the chicken cTNT MSE1-4 RNA contains an alternative exon flanked by four MSEs. Below, FIG. 9A shows the results of the UV-cross-linking assays, wherein GST-MBNL1 bound weakly to MSE1 and strongly to MSE4.

FIG. 9B shows UV-cross-linking assay results for competition of GST-MBNL1 binding to labeled chicken cTNT MSE1-4 RNA by non-labeled MSE RNAs. Picomoles of competitor RNA are indicated.

FIG. 9C shows the results of the scanning mutagenesis performed, identifying two MBNL1-binding sites within MSE4.

FIG. 9D shows an alignment of the four MBNL1-binding motifs in human and chicken cTNT, which reveals a common motif.

FIG. 10A shows, in bar graph form, the results of the over-expression and depletion experiments with respect to the wild-type cTNT minigene, co-transfected with the indicated siRNA constructs, a plasmid expressing a DMPK minigene with 960 CUG repeats (Philips et al, 1998) or a GFP-MBNL1 expression plasmid in HeLa cells.

FIG. 10B shows the results with respect to the mutant cTNT minigene with point mutations that prevent CUG-BP1 binding and regulation.

FIG. 11A shows, in bar graph form, the results of the over-expression and depletion experiments with respect to the mutant human IR minigene lacking the CUG-BP1-binding site in HEK293 cells.

FIG. 11B shows the results with respect to the human IR minigene lacking the CUG-BP1-binding site.

FIG. 12 shows the results of FISH (left panels) and IF (middle panels) analyses of frozen sections of DM1 brain showing nuclear foci of mutant DMPK mRNA. FISH, IF, and nuclear stain (DAPI, blue) images are merged in panels on the right. In (A), FISH (without IF) using Texas Red-labeled CAG repeat probe shows an RNA inclusion in frontal cortical neuron. Autofluorescence from lipofuscin occurs at broad spectrum of wavelengths. It appears in every color channel and as yellow-brown perinuclear material in the merged image. RNA inclusions in cerebral cortex are confined to neurons identified by IF for NeuN (B) or MAP2 (C). Small foci are present in cerebellar Purkinje cells (D) or oligodendrocytes of the centrum semiovale (E) identified by IF for calbindin or CNPase, respectively. (F) RNA foci do not colocalize with PML bodies in cortical neurons. Bar, 5 μm, applies to all panels.

FIG. 13 shows RNA foci in dentate gyrus and subcortical neurons in DM1, as visualized by FISH and IF analysis. FISH (CAG repeat probe, red) merged with IF (anti-NeuN antibody, green) and nuclear stain (DAPI, blue). SN, substantia nigra. Bar, 5 μm, applies to all panels.

FIG. 14(A) shows foci of mutant RNA in neuronal and muscle nuclei, as visualized by FISH and IF analysis. Processing was carried out on the same slide and imaging under the same exposure settings. (B) depicts, in bar graph form, fluorescence area×intensity of RNA foci in paired samples of frontal cortex and skeletal muscle from the same patient; n=3 patients, 20 nuclei per sample (p<10 −10 ).

FIG. 15 shows the results of FISH and IF analyses of sections of temporal or frontal cortical neurons showing colocalization of mutant DMPK mRNA [(CUG)n] with 20Sa subunit of proteasome (A), MBNL2 (E), and hnRNP F (F). There is a marked redistribution of MBNL1 into RNA foci in DM1 cortical neurons (G), compared to the distribution in the nucleus (excluding nucleolus) and cytoplasm of normal neurons (H). Mutant DMPK mRNA does not colocalize with the PM/Scl100 (nuclear) component of the exosome (B), CUGBP1 (C), or NF90 (D). RARγ does not colocalize with RNA foci in DM1 cortical neurons (I). The distribution of RARγ in the DM1 (I) and non-neurologic-disease (J) neuronal nucleus is similar. Bar, 5 μm, applies to all panels.

FIG. 16 shows the results of FISH analysis combined with IF analysis of sections of DM1 temporal or frontal cortex. FISH (CAG repeat probe, red, left panels) and IF (middle panels, antibody to indicated protein, green) are merged with nuclear stain (DAPI, blue) in right panels. CUG expansion RNA colocalizes with proteasome 11Sγ subunit (A) and hnRNP H (C) but not with double-stranded RNA binding protein ADAR1 (B), hnRNP M (D), or Sp1 (E). Bar, 5 μm, applies to all panels.

FIG. 17 depicts, in graph form, immunofluorescence (area×intensity) for MBNL1 in the nucleus, excluding nucleolus and RNA foci, as determined for 20 neurons in sections of temporal cortex from 3 individuals with DM1 and 3 controls without neurologic disease (C).

FIG. 18 shows the regulation of alternative splicing of the NMDA NR1 receptor (NMDAR1), amyloid beta precursor protein (APP), and microtubule-associated protein tau (MAPT) in DM1. (A) shows splice products obtained by RT-PCR amplification of RNA isolated from non-disease control (n=5) or DM1 (n=7) temporal cortex. Exon utilization for each splice product is shown in diagram. (B) provides quantification of RT-PCR splicing assay (triplicates). ex, exon.

Muscleblind Proteins

Proteins in the muscleblind-like (MBNL) family bind to expanded CUG repeats in vitro and colocalize with mutant DM and HSA LR transcripts in vivo. Human muscleblind genes MBNL1 (SEQ ID NO: 1), MBNL2 (SEQ ID NO: 2), and MBNL3 (SEQ ID NO: 3) are homologous to the Drosophila gene muscleblind, which is essential for muscle and eye differentiation. MBNL1, the major MBNL gene expressed in human skeletal muscle, encodes multiple protein isoforms, including some that bind to expanded CUG repeats (41 to 42 kD) and others that fail to bind (31 kD isoform), generated by exon 3 skipping.

In fact, MBNL1 was identified in HeLa cells based on its ability to bind double-stranded CUG repeats (Miller J W, Urbinati C R, Teng-Umnuay P, Stenberg M G, Byrne B J, Thornton C A, Swanson M S (2000), EMBO J 19: 4439-4448). All three MBNL gene products colocalize with the expanded repeat RNA foci in vivo (Fardaei M, Rogers M T, Thorpe H M, Larkin K, Hamshere M G, Harper P S, Brook J D (2002), Hum Mol Genet 11: 805-814). Loss of MBNL function due to sequestration on CUG repeat RNA is proposed to play a role in DM pathogenesis (Miller J W, Urbinati C R, Teng-Umnuay P, Stenberg M G, Byrne B J, Thornton C A, Swanson M S (2000), EMBO J 19: 4439-4448). Thus, while expression of CUG and CCUG expansion RNAs induces MBNL recruitment into nuclear RNA foci, there is no evidence that this relocalization results in muscleblind depletion and functional impairment.

Recombinant Adeno-Associated Vectors

Recombinant AAV (rAAV) vectors have been used for expressing gene products in animals, see, for example, U.S. Pat. No. 5,193,941 and WO 94/13788. Other patents and publications describe AAV vectors and uses, the uses generally being related to expression of gene products either in vitro (usually tissue cultures) or in vivo (usually in the lungs or oral mucosa, the normal sites of AAV infection, but expression in other tissues, such as the central nervous system and in cardiac tissue has been observed).

AAV vectors have certain advantages over other well-characterized vector systems. First, like adenovirus, AAV infects non-dividing cells. Second, all the AAV viral genes are eliminated in the vector. Since the viral gene expression-induced immune reaction is no longer a concern, AAV vectors are safer than adenovirus vectors. As AAV is an integration virus, integration into the host chromosome will maintain the transgene in the cells. AAV is an extremely stable virus, resistant to many detergents, pH changes and heat (stable at 56° C. for about an hour). AAV can be lyophilized and redissolved without losing significant activity. Finally, AAV causes no known diseases or pathogenic symptoms in humans. Therefore, AAV is a very promising delivery vehicle for gene therapy.

Transduction of rAAV vectors harboring the bacterial β-galactosidase gene by single injection into the quadriceps of mice demonstrated that expression was maintained long-term and the expression did not decrease substantially during that time (Xiao et al., J. Virol., 70:8098-8108 (1996)). Other targets successfully transduced with rAAV vectors include: T-lymphocytes and B-lymphocytes, human erythroleukemia cells, different regions of the rat brain, the striatum of the rat brain in a Parkinson's Disease model with the tyrosine hydroxylase gene, heart of the pig and rat with the LacZ gene, the peripheral auditory system of the guinea pig and bronchial epithelia of the rabbit and monkey.

In one embodiment, the invention provides a vector for effective expression of a protein with MBNL1, MBNL2, or MBNL3 (or combinations thereof) function to treat conditions associated with aberrant microsatellite expansions. In an additional embodiment, the vector of the invention is recombinant adeno-associated virus (rAAV) vectors. In one embodiment of the invention, the rAAV contains a transgene that expresses an MBNL1, MBNL2, or MBNL3 (or combinations thereof) protein. In an additional embodiment, the invention provides a rAAV containing a transgene that expresses MBNL1 (for example, the 41 kD isoform).

Isolation of the DNA encoding MBNL polypeptides allows one to use methods well-known to the person of ordinary skill in the art to make changes in the codons for specific amino acids such that the codons are “preferred usage” codons for a given species.

In one embodiment, the rAAV of the invention includes a promoter, which directs the initiation of RNA transcription in the cell of interest. The promoter may be constitutive or regulated. Regulated promoters include inducible promoters and repressible promoters. In an additional embodiment of the invention, the regulation of the promoter is associated with an “operator”, to which an inducer or repressor binds. The promoter may be a “ubiquitous” promoter active in essentially all cells of the host organism or may be a promoter with expression more or less specific to the target cells. Known strong promoters that find common use to obtain high levels of recombinant protein expression include the herpes simplex thymidine kinase promoter, SV40 promoter and LTRs such as that obtained from Moloney leukemia retrovirus. For the gene to be expressed, the coding sequence must be operably linked to a promoter sequence functional in the target cell.

It is not necessary that the AAV-derived sequences correspond exactly with wild-type AAV prototypes. For example, in one embodiment, the rAAV vectors of the invention may feature modified inverted terminal repeats and other sequences, provided that the rAAV vectors can replicate and be packaged with the assistance of helper virus, and establish a nonpathogenic latent infection in target cells. Typically, because of the packaging limitations of AAV, the polynucleotides encoding MBNL1 domain sequences and the regulatory elements can have a length of up to about 5,500 bases.

Numerous applications of the present invention, e.g., making transgenic constructs, involve the cloning, synthesis, maintenance, mutagenesis, and other manipulations of nucleic acid sequences that can be performed using routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).

For propagation of the rAAV vectors in vitro, susceptible cells are co-transfected with an AAV-derived vector DNA and a suitable AAV-derived helper virus or plasmid harboring the AAV rep gene, AAV cap gene or both and infected by a helper virus, including herpesvirus, adenovirus or a suitable non-AAV helper plasmid using any number of transfection methods, including, inter alia, calcium-phosphate transfection, lipofection or other techniques known to those skilled in the art. The ratio of helper plasmids to the quantity of vector plasmid containing the gene of interest range from 1:1-1:10. This procedure produces recombinant AAV vectors; the vector plasmid contains the recombinant AAV genome flanked by the AAV ITRs. The AAV-derived helper virus or helper plasmid may be any virus or plasmid which is capable, on expression of the AAV genes it carries, of providing proteins necessary for the replication and packaging of the rAAV vector in a suitable host cell, for the purpose of producing rAAV vector stock.

In one embodiment, the target cells of the rAAV vectors of the invention are cells capable of expressing polypeptides with MBNL1 activity. In another embodiment of the invention, the cells are normal cells cultured in vitro. In further embodiments, the target cells of the rAAV vectors of the invention are human cells, or cells of other mammals, such as nonhuman primates and mammals of the orders Rodenta (mice, rats, rabbit and hamsters), Carnivora (cats and dogs) and Arteriodactyla (cows, pigs, sheep, goats and horses). In one embodiment of the invention, the cells are part of a living mammal at the time the rAAV vectors are delivered to the cell. The mammal may be at any stage of development at the time of delivery, e.g., embryonic, fetal, infantile, juvenile or adult. Additionally, the cells may be healthy or diseased.

In one embodiment, the rAAV vectors of the invention may be administered as viral particles alone, whether as an in vivo direct delivery to the vasculature or as an ex vivo treatment comprising administering the rAAV vector viral particles in vitro to cells from the animal receiving treatment followed by introduction of the transduced cells back into the donor. Alternatively, the rAAV vector virus particles can be used to transduce cells in conjunction with secondary agents known to enhance the efficiency of transduction, see, e.g., WO Ser. No. 95/33824 for a variety of secondary agents. The effective amount of rAAV vectors to be administered will vary from patient to patient. Accordingly, effective amounts are best determined by the physician administering the rAAV vectors, and appropriate dosages can be determined readily by one of ordinary skill in the art.

In one embodiment, the rAAV construct of the invention expresses human MBNL1 (rAAV-MBNL1 (rAAV-MBNL1/41)). In an additional embodiment of the invention, injection of the rAAV-MBNL1/41 into the tibialis anterior (TA) muscles of a transgenic model for DM that expresses a human skeletal α-actin transgene carrying 250 CTG repeats (HSA LR —a mouse model which develops myotonia and muscle degeneration similar to muscle abnormalities seen in DM patients) results in a functional reversal of a DM-related phenotype, namely, reversal of mis-splicing of the Clcn1 skeletal muscle chloride channel, which results in myotonia.

In one embodiment, the invention is directed to methods for treating or preventing various disorders and conditions associated with aberrant microsatellite expansions in a mammal, said method comprising administering to the mammal a therapeutically effective amount of rAAV containing a transgene that encodes a protein selected from the group consisting of MBNL1, MBNL2, MBNL3, and combinations thereof. In a further embodiment of the invention, the protein is MBNL1. In another embodiment of the invention, the mammal is a human. In another embodiment, the transgene is human. In one embodiment of the invention, the disease associated with aberrant microsatellite expansion is a neurological or neuromuscular disease. In an additional embodiment of the invention, the disease is myotonic dystrophy. In yet another embodiment of the invention, the disease is SCA8.

In additional embodiments, the present invention provides methods for treating or preventing a disease or condition related to any physiological process affected by MBNL1, said method comprising administering to the mammal a therapeutically effective amount of rAAV containing a transgene that expresses the MBNL1 protein.

In one embodiment, the invention is directed to methods for treating or preventing various disorders and conditions associated with aberrant microsatellite expansions in a mammal, said method comprising administering to the mammal a therapeutically effective amount of rAAV containing a transgene that encodes a protein selected from the group consisting of MBNL1, MBNL2, MBNL3, and combinations thereof, wherein treating comprises reversing the mis-splicing of the Clcn1 skeletal muscle chloride channel.

In another embodiment of the invention, treating comprises reversing the mis-splicing of the Amyloid beta (A4) precursor protein (APP). The mis-splicing may correspond to alternative splicing of exon 7. In another embodiment of the invention, treating comprises reversing the mis-splicing of the NMDA receptor NR1 (GRIN1). The mis-splicing may correspond to alternative splicing of exon 5. In yet another embodiment of the invention, treating comprises reversing the mis-splicing of the Microtubule-associated protein tau (MAPT). The mis-splicing may correspond to alternative splicing of exon 2. In yet another embodiment of the invention, treating comprises reversing the mis-splicing of the TNNT2 (cTNT). The mis-splicing may correspond to alternative splicing of exon 5.

In one embodiment, the invention is directed to methods for treating or preventing various disorders and conditions associated with aberrant microsatellite expansions in a mammal, said method comprising administering to the mammal a therapeutically effective amount of rAAV containing a transgene that encodes a protein selected from the group consisting of MBNL1, MBNL2, MBNL3, and combinations thereof, wherein the mammal has RNA inclusions in neuronal cells.

One embodiment of the invention is directed to a pharmaceutical composition comprising rAAV containing a transgene that encodes at least one protein selected from the group consisting of MBNL1, MBNL2, MBNL3, and combinations thereof. In one embodiment of the invention, the protein is MBNL1. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17.sup.th ed. 1985).

Formulations for both ex vivo and in vivo administrations include suspensions in liquid or emulsified liquids. The active ingredient (rAAV vector) is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances, such as, wetting or emulsifying agents, pH buffering agents, stabilizing agents or other reagents that enhance the effectiveness of the pharmaceutical composition.

Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, orally, nasally, topically, intravenously, intraperitoneally, intravesically or intrathecally. The formulations of compounds can be presented in unit-dose or multi-dose scaled containers, such as ampules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. The modulators can also be administered as part a of prepared food or drug.

The dose administered to a patient, in the context of the present invention is often varied to assess the effect of various concentrations of a compound on a transgenic animal. The dose will also be determined by, e.g., the body weight or surface area of the area to be exposed to the compound. In general, the dose equivalent of a modulator is from about 1 ng/kg to 10 mg/kg for a typical subject. Administration can be accomplished via single or divided doses.

Pharmaceutical preparations of the disclosed gene vectors may be administered intravenously, parenterally or intraperitoneally. Solutions of pharmaceutically acceptable salts can be prepared in water suitable mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations will contain a preservative to prevent growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, such as, parabens, chlotobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

Solutions of the AAV vector as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. A dispersion of AAV particles also can be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, the preparations contain a preservative to prevent the growth of microorganisms. The sterile aqueous media employed are obtainable by standard techniques well known to those skilled in the art.

Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The composition can be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organics acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.

For parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.

The pharmaceutical forms suitable for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that parenteral administration is possible. The formulation must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile parenteral formulations are prepared by incorporating the AAV vector in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying which yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

The rAAV containing a transgene that expresses the MBNL1 protein may, for example, be prepared by: culturing a composition comprising cells transiently transfected with an AAV helper plasmid comprising AAV rep and cap nucleic acid sequences encoding AAV rep and cap proteins, an adenoviral helper plasmid comprising essential adenovirus helper genes selected from the group consisting of E1A, E1B, E2A, E4, E4O RF6, E4O RF6/7, VA, and combinations thereof, and an AAV vector comprising first and second AAV ITRs flanking a DNA sequence encoding MBNL1 polypeptide, said sequence being operably linked to a promoter DNA sequence, in the absence of adenovirus particles and under conditions suitable for production of recombinant AAV, and purifying rAAV therefrom.

In one embodiment, the invention is directed to a transgenic animal having a substantial deletion of one or more MBNL1 exon(s). The transgenic animals of the invention can be any mammal other than humans. In one embodiment, the mammal is a rodent. In another embodiment of the invention, the rodent is a mouse. An additional embodiment is directed to a cell isolated from the transgenic animal of the invention.

In a further embodiment, the transgenic animal of the invention has a substantial deletion of Mbnl1 exon 3. In one embodiment, Mbnl1 exon 3 in the transgenic animal of the invention is deleted in its entirety.

In one embodiment of the invention, the transgenic mouse having a substantial deletion of Mbnl1 exon 3 constitutes an animal model for microsatellite expansion disease in mammals. In another embodiment of the invention, the mammal is a primate. In yet another embodiment of the invention, the primate is a human.

In one embodiment of the invention, the microsatellite expansion disease is caused by a microsatellite expansion in a coding region of DNA. In another embodiment of the invention, the microsatellite expansion disease is caused by a microsatellite expansion in a non-coding region of DNA. In one embodiment of the invention, the disease associated with aberrant expansion of microsatellites is myotonic dystrophy. Accordingly, in one embodiment of the invention, the mouse Mbnl1 gene knockout model exhibits myotonia and ocular cataracts.

In one embodiment, the invention is directed to a mouse model for disease associated with aberrant microsatellite expansion, comprising a mouse having a substantial deletion of Mbnl1 exon 3 (E3) in the mouse genome, wherein said mouse exhibits symptoms typical of a disease associated with aberrant microsatellite expansion in humans, wherein said mouse has loss of functional ClC-1 protein. In another embodiment of the invention, mouse has loss of functional Amyloid beta (A4) precursor protein. In another embodiment of the invention, the mouse has loss of functional NMDA receptor NR1. In yet another embodiment of the invention, the mouse has loss of functional Microtubule-associated protein tau. In another embodiment of the invention, the mouse has loss of functional TNNT2 protein. In another embodiment of the invention, the mouse has loss of functional TNNT3 protein.

One embodiment is directed to methods for preparing the transgenic animals of the invention. The transgenic animal of the invention may, for example, be prepared by transfecting a plurality of mouse embryonic stem cells with a nucleic acid comprising an MBNL1 gene with a substantial deletion of exon 3, selecting for transgenic embryonic stem cells having incorporated said nucleic acid into their genome, introducing at least one of said transgenic embryonic stem cells into an embryo to produce a chimeric mouse comprising at least one of said transgenic embryonic stem cells, breeding said chimeric mouse with a wild-type mouse to obtain F1 progeny heterozygous for the MBNL1 gene with a deletion of exon 3, and breeding a male mouse of said F1 progeny with a female mouse of said F1 progeny to obtain F2 progeny homozygous for MBNL1 gene with a deletion of exon 3, wherein the said mouse exhibits a phenotype indicative disease associated with aberrant microsatellite expansion, for example, myotonic dystrophy.

Additional embodiments are directed to methods for using the transgenic animals of the invention as animal models to study MBNL1 function in vivo, and for evaluating side effects of MBNL1-inhibiting compounds. For example, if a compound known to inhibit MBNL1 is administered to an MBNL1 knockout mouse, any detected effects of the compound on the mouse can be concluded to be MBNL1-independent.

In further embodiments, the transgenic mammals of the invention, and cells thereof, can be used as animal models to identify compounds useful in the treatment of diseases associated with aberrant microsatellite expansions (such as, in one embodiment, myotonic dystrophy) and to assess the functional effect of a test compound on cells or animals afflicted with such disease. Such compounds can be any small chemical compound, including polypeptides, polynucleotides, amino acids, nucleotides, carbohydrates, lipids, or any other organic or inorganic molecule. Alternatively, the compounds can be genetically altered versions of the MBNL1 gene.

In one embodiment of the invention, assessing the effects of a compound on cells or animals, e.g., the transgenic animals of the invention having a substantial deletion of MBNL1 exon 3, involves providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator or binding compounds). Such “combinatorial chemical libraries” are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display as desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds” or can themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical “building blocks” such as reagents. Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37:487-493 and Houghton, et al. (1991) Nature, 354:84-88).

To assess the effect of a compound on an animal, or to treat or prevent a condition associated with aberrant microsatellite expansion, for example, myotonic dystrophy, in an animal, administration of the compound can be achieved by any of the routes normally used for introducing a compound into ultimate contact with the tissue to be treated. The compounds are administered in any suitable manner, optionally with pharmaceutically acceptable carriers. Suitable methods of administering such compounds are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.

Although MBNL1 is referred to in the individual descriptions of the embodiments of the invention, MBNL2 and MBNL3 may likewise be contemplated for each embodiment.

A “transgene” refers to genetic material that is introduced, or is capable of being introduced, into cells of a host animal. Typically, once a “transgene” is introduced into the cells of the host animal, it is maintained, either transiently or permanently, by, e.g., insertion into the host genome. In preferred embodiments of the present invention, a transgene is inserted into the host genome by homologous recombination, thereby replacing the endogenous gene with the transgene. Often, a transgene contains a coding sequence, operably linked to a promoter, that encodes a protein, e.g., a marker protein that allows the detection of the transgene in the cell. “Transgenic” refers to any cell or organism that comprises a transgene.

A “host” animal or mammal refers to any animal that is used to practice the herein-described methods, i.e. animals into which a transgene is introduced to disrupt an endogenous MBNL1 gene. For use in the present invention, such animals include any non-human mammals including, but not limited to, mice, rats, rabbits, and hamsters.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term nucleic acid is used interchangeably with gene, cDNA and nucleotide.

The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.

The term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

The term “heterologous” when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

A “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.”

The term “immunoassay” is an assay that uses an antibody to specifically bind an antigen. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.

The phrase “specifically (or selectively) binds” to an antibody or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein For example, polyclonal antibodies raised to an MBNL1 polypeptide from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the MBNL1 protein and not with other proteins, except for polymorphic variants and alleles of the MBNL1 protein.

The term “transduction” refers to the introduction of foreign DNA into cells of an organism (in vivo).

The term “transfection” refers to the introduction of foreign DNA into cells in culture (in vitro). Genetic modification of eukaryotic cells by introduction of foreign DNA using chemical means. In transient transfection, expression occurs from unintegrated foreign DNA and can be detected for a few days after transfection.

The term “titer” refers to the number of virus particles produced per ml. The assay system to determine the number of virus particles produced varies considerably depending on the virus in question. High titers are generally essential for successful gene therapy since they allow introduction of the therapeutic gene carried by the virus into the maximum number of cells.

The terms “treating” and “treatment” as used herein include any treatment of a condition or disease in a subject, and include inhibiting the disease or condition, (i.e. arresting its development), relieving the disease or condition (i.e. causing some degree of regression of the condition or delaying progression in the disease), or relieving (to some degree) the conditions caused by the disease (i.e. symptoms of the disease).

The term “vector” refers to a vehicle, usually a biological entity, such as a virus, used for the delivery of genes into an organism. A reagent that facilitates the introduction of foreign genes into cells.

The phrase “packaging cells” refers to cells that have been transfected with plasmids containing the cap and rep genes from AAV.

As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically-active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.

A “substantial” deletion of exon 3 signifies a deletion extensive enough to lend to the phenotype indicative of a disease associated with aberrant microsatellite expansion.

Use of the terms “an”, “a” and “the” and similar terms used in claiming or describing the invention are intended to be construed as including both the singular and plural, unless clearly otherwise indicated or contraindicated. The terms “including”, “having” and “containing” are to be construed as open-ended in the same manner as the terms “comprising” or “comprises” are commonly accepted as including but not limiting to the explicitly set forth subject matter. The term “comprising” and the like are construed to encompass the phrases “consisting of” and “consisting essentially of”.

The methods and processes described herein may be performed in any suitable order unless otherwise indicated or clearly rendered inoperable by a modification in order.

Limited and narrow interpretation of descriptive language intended to better illustrate the invention is not to be construed as limiting in any way nor to limit the scope of the invention contemplated by the inventors.

The invention, now described generally and in some detail, will be understood more readily by reference to the following examples, which are provided by way of reference and are in no manner intended to limit the scope of what the inventors regard as their invention.